*2.5. PCR-Amplification of Targeting Constructs*

For gene disruption in *L. braziliensis*, PCR amplification of sgRNA templates (using a common sgRNA scaffold primer) and of donor DNAs, the latter from pTBlast and pTPuro plasmids [17], was done using the ExpandTM High Fidelity PCR System (Roche, Mannheim, Germany) and PCR conditions as described [40].

For gene disruption in *L. major*, sgRNA templates were amplified in a total volume of 20 µL using 1 × iProof high-fidelity PCR master mix (Bio-Rad, München, Germany), 2 µM G00 primer (sgRNA scaffold) and 2 µM LmHSP23-specific 3'sgRNA or 5'sgRNA primer (Table S1). Cycling conditions were 30 s at 98 ◦C followed by 35 cycles of 10 s at 98 ◦C, 30 s at 55 ◦C, 15 s at 72 ◦C, and a final elongation step of 10 min at 72 ◦C. The targeting fragments were amplified from 10 ng pTPuro or pTBlast plasmid in 1 × iProof mix (Bio-Rad) using 2 µM forward and reverse primers, 3% DMSO in a total volume of 25 µL. PCR steps were 3 min at 98 ◦C followed by 35 cycles of 30 s at 98 ◦C, 30 s at 65 ◦C, 30 s at 72 ◦C, and a final elongation step of 5 min at 72 ◦C.
